U.S. patent application number 12/465572 was filed with the patent office on 2010-11-18 for human nt-pro b-type natriuretic peptide assay having reduced cross-reactivity with other peptide forms.
This patent application is currently assigned to Abbott Laboratories. Invention is credited to John George Konrath, Jeffrey Allen Moore.
Application Number | 20100291709 12/465572 |
Document ID | / |
Family ID | 42263924 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291709 |
Kind Code |
A1 |
Konrath; John George ; et
al. |
November 18, 2010 |
HUMAN NT-PRO B-TYPE NATRIURETIC PEPTIDE ASSAY HAVING REDUCED
CROSS-REACTIVITY WITH OTHER PEPTIDE FORMS
Abstract
The present disclosure relates to assays for detecting and/or
quantifying the amount of human NT-pro B-type natriuretic peptide
or human NT-pro B-type natriuretic peptide fragment in a test
sample.
Inventors: |
Konrath; John George; (Lake
Villa, IL) ; Moore; Jeffrey Allen; (Gurnee,
IL) |
Correspondence
Address: |
PAUL D. YASGER;ABBOTT LABORATORIES
100 ABBOTT PARK ROAD, DEPT. 377/AP6A
ABBOTT PARK
IL
60064-6008
US
|
Assignee: |
Abbott Laboratories
Abbott Park
IL
|
Family ID: |
42263924 |
Appl. No.: |
12/465572 |
Filed: |
May 13, 2009 |
Current U.S.
Class: |
436/518 ;
530/387.1 |
Current CPC
Class: |
G01N 33/74 20130101;
C07K 16/26 20130101; G01N 2333/58 20130101 |
Class at
Publication: |
436/518 ;
530/387.1 |
International
Class: |
G01N 33/543 20060101
G01N033/543; C07K 16/00 20060101 C07K016/00 |
Claims
1. An immunoassay for quantifying the amount of human NT-pro B-type
natriuretic peptide ("human NT-proBNP") in a test sample being
tested for or suspected of containing human NT-proBNP, the
immunoassay having reduced cross-reactivity with any human pro
B-type natriuretic peptide ("human proBNP") present in the test
sample and comprising the steps of: (a) contacting at least one
capture antibody that binds to human NT-proBNP and that has been
immobilized onto a solid phase to produce an immobilized antibody
with said test sample to form a first mixture comprising an at
least one capture antibody-human NT-proBNP complex, wherein said
capture antibody comprises antibody 15F11; (b) contacting said
first mixture comprising the at least one capture antibody-human
NT-proBNP complex with at least one detection antibody that binds
to human NT-proBNP and that has been conjugated to a detectable
label to form a second mixture comprising at least one capture
antibody-human NT-proBNP-at least one detection antibody complex,
wherein the detection antibody comprises antibody 24E11 or 15C4;
and (c) determining the amount of the at least one capture
antibody-human NT-proBNP-at least one detection antibody complex
formed in step (b) by detecting the detectable label as a measure
of the amount of human NT-proBNP contained in the test sample,
wherein the at least one capture antibody and the at least one
detection antibody, when used together, exhibit a cross-reactivity
of less than about 1.0% with any human proBNP present in the test
sample.
2. The immunoassay of claim 1, wherein the at least one detection
antibody is 24E11.
3. The immunoassay of claim 1, wherein the at least one detection
antibody is 15C4.
4. An immunoassay for quantifying the amount of human NT-pro B-type
natriuretic peptide ("human NT-proBNP") in a test sample being
tested for or suspected of containing human NT-proBNP, the
immunoassay having reduced cross-reactivity with any human pro
B-type natriuretic peptide ("human proBNP") present in the test
sample and comprising the steps of: (a) contacting said test sample
with at least one detection antibody that binds to human NT-proBNP
and that has been conjugated to a detectable label to form a first
mixture comprising an at least one human NT-proBNP-detection
antibody complex, wherein the detection antibody comprises antibody
24E11 or 15C4; (b) contacting said first mixture comprising said at
least one human NT-proBNP-detection antibody complex with at least
one capture antibody that binds to human NT-proBNP and that has
been immobilized on to a solid phase to produce an immobilized
antibody to form a second mixture comprising an at least one
capture antibody-human NT-proBNP-at least one detection antibody
complex, wherein said at least one capture antibody comprises
antibody 15F11; and (c) determining the amount of the at least one
capture antibody-human NT-proBNP-at least one detection antibody
complex formed in step (b) by detecting the detectable label as a
measure of the amount of human NT-proBNP contained in the test
sample, wherein the at least one capture antibody and the at least
one detection antibody, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP
present in the test sample.
5. The immunoassay of claim 4, wherein the at least one detection
antibody is 24E11.
6. The immunoassay of claim 4, wherein the at least one detection
antibody is 15C4.
7. An immunoassay for quantifying the amount of human NT-pro B-type
natriuretic peptide ("human NT-proBNP") in a test sample being
tested for or suspected of containing human NT-proBNP, the
immunoassay having reduced cross-reactivity with any human pro
B-type natriuretic peptide ("human proBNP") present in the test
sample and comprising the steps of: (a) contacting a test sample
with at least one capture antibody that binds to human NT-proBNP
and that has been immobilized onto a solid phase to produce an
immobilized antibody and with at least one detection antibody that
binds to human NT-proBNP and that has been conjugated to a
detectable label to form an at least one capture antibody-human
NT-proBNP-at least one detection antibody complex, wherein the at
least one capture antibody comprises antibody 15F11 and the at
least one detection antibody is antibody 24E11 or 15C4; and (b)
determining the amount of the at least one capture antibody-human
NT-proBNP-- at least one detection antibody complex formed in step
(a) by detecting the detectable label as a measure of the amount of
human NT-proBNP contained in the test sample, wherein the at least
one capture antibody and the at least one second antibody
conjugated to the detectable label, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP
present in the test sample.
8. The immunoassay of claim 7, wherein the at least one detection
antibody is 24E11.
9. The immunoassay of claim 7, wherein the at least one detection
antibody is 15C4.
10. An immunodiagnostic reagent comprising at least one capture
antibody and at least one detection antibody specific for human
NT-pro B-type natriuretic peptide ("human NT-proBNP"), and that,
when used together, exhibit a cross-reactivity of less than about
1.0% with any human pro B-type natriuretic peptide ("human
proBNP").
11. The immunodiagnostic reagent of claim 10, wherein the capture
antibody is antibody 15F11 and the detection antibody is antibody
24E11 or 15C4.
12. A kit for the detection of human NT-pro B-type natriuretic
peptide ("human NT-proBNP") in a test sample, said kit comprising:
(a) instructions for conducting the assay of the test sample; and
(b) an immunodiagnostic reagent that comprises at least one capture
antibody and at least one detection antibody specific for human
NT-pro B-type natriuretic peptide ("human NT-proBNP"), and that,
when used together, exhibit a cross-reactivity of less than about
1.0% with any human pro B-type natriuretic peptide ("human
proBNP").
13. The kit of claim 12, wherein capture antibody is antibody 15F11
and the detection antibody is antibody 24E11 or 15C4.
Description
RELATED APPLICATION INFORMATION
[0001] None.
TECHNICAL FIELD
[0002] The present disclosure relates to assays for detecting
and/or quantifying the amount of human NT-pro B-type natriuretic
peptide or human NT-pro B-type natriuretic peptide fragment in a
test sample. Specifically, the assays of the present disclosure
exhibit less than about one percent (1.0%) cross-reactivity with
any human pro B-type natriuretic peptide present or contained in a
test sample.
BACKGROUND
[0003] Atrial natriuretic peptide (hereinafter "ANP"), B-type
natriuretic peptide (hereinafter "BNP"), C-type natriuretic peptide
(hereinafter "CNP") and Dendroaspis natriuretic peptide
(hereinafter "DNP") are each members of a family of hormones known
as "natriuretic peptides". ANP and BNP share a wide spectrum of
biological properties and belong to the cardiac natriuretic system.
Both ANP and BNP are of myocardial cell origin while CNP is of
endothelial cell origin. DNP was isolated from the venom of the
green mamba snake and possesses structural similarity to ANP, BNP
and CNP.
[0004] ANP is secreted by the heart in the atria. ANP has a 17
amino acid ring closed by a disulfide bond between two cysteine
residues. Eleven of the seventeen amino acids in the ring are
conserved across ANP, BNP, CNP and DNP. In addition to the 17 amino
acid ring structure, ANP has an amino-terminal tail of 6 amino
acids and a carboxy-terminal tail of 5 amino acids. ANP is produced
as a 126 amino acid pro-ANP form that is the major storage form of
ANP. After proteolytic cleavage between amino acids 98 and 99, the
mature 28 amino acid peptide ANP is found in coronary sinus plasma
(See Yandle, J. Internal Med., 235:561-576 (1994)).
[0005] BNP received its name because it was first isolated from
porcine brain, thus, initially, "BNP" stood for "brain natriuretic
peptide". However, because BNP was found to belong to the cardiac
natriuretic system, the word "brain" was changed to "B-type".
Therefore, "BNP" now refers to "B-type natriuretic peptide". In
humans, BNP is secreted by the heart through the coronary sinus,
predominantly from the cardiac ventricles. The pre-pro peptide
precursor of human BNP (hereinafter "human pre-proBNP") is 134
amino acids in length (SEQ ID NO:1) and comprises a short signal
peptide, which is enzymatically cleaved off to release the human
pro peptide of BNP (hereinafter "human proBNP") which is 108 amino
acids in length (SEQ ID NO:2). Human proBNP is further cleaved into
an N-terminal pro peptide of human BNP (hereinafter "human
NT-proBNP") which is 76 amino acids in length (SEQ ID NO:3) and the
active hormone, human BNP (hereinafter "hBNP" or "hBNP-32"), which
is 32 amino acids in length (SEQ ID NO:4). It has been suggested
that each of human NT pro-BNP, hBNP-32, and human proBNP--can
circulate in human plasma (See, Tateyama et al., Biochem. Biophys.
Res. Commun. 185: 760-7 (1992); Hunt et al., Biochem. Biophys. Res.
Commun. 214: 1175-83 (1995)).
[0006] CNP was first found in the brain, however, most of it
originates in endothelial and renal cells. It is widely distributed
in the vasculature, brain, bone and endothelium. Little if any CNP
is present in the heart. Pro-CNP is a 103 amino acid peptide that
is processed into either CNP-53 (amino acids 51 to 103) or CNP-22
(amino acids 82 to 103) that are the active peptides. Like ANP, CNP
has a 17 amino acid ring closed by a disulfide bond between
cysteine residues. In addition to this 17 amino acid ring
structure, CNP-22 has an amino-terminal tail of 5 amino acids and
contains no carboxy-terminal tail. CNP-53 is identical to CNP-22
except for a 31 amino acid extension at the amino terminal end.
[0007] As mentioned previously, DNP was isolated from the venom of
the green mamba snake. The mature form of DNP is made up of 38
amino acids. DNP-like immunoreactivity (DNP-LI) has been reported
in human plasma and the plasma concentration of DNP-LI has been
found to be elevated in patients with congestive heart failure
(See, Cataliotti, et al., Mayo Clin. Proc., 76:111-1119 (2001)).
Additionally, it is also known that the infusion of synthetic DNP
results in marked natriuresis and diuresis in association with
increased plasma and urinary cyclic guanosine monophosphate.
Id.
[0008] In humans, heart disease can stimulate the secretion of ANP
and BNP. In fact, the secretion of ANP and BNP in humans typically
reflects a change in cardiac function. Specifically, the secretion
of ANP is typically accelerated when the atrium undergoes a load,
while the biosynthesis and secretion of BNP is stimulated when the
ventricle undergoes a load. Thereupon, both ANP and BNP are useful
as indicators in the diagnosis of heart disease. However, despite
this and over time, BNP has become recognized as a useful indicator
in the diagnosis of heart disease, more so than ANP. For example,
the blood concentration of BNP is only 1/6 of ANP in a normal
subject but it becomes higher than ANP in patients of heart
failure. Moreover, the blood concentration of BNP increases in the
case of heart failure like ANP, and the plasma concentration of BNP
often exceeds that of ANP, thus reflecting more accurately the
severity of heart dysfunction. Moreover, BNP level in patients of
heart failure sometimes increases to several tens times to several
hundreds times of that of healthy normal subjects.
[0009] It is known that human proBNP, human NT-proBNP and hBNP can
circulate and may be detected in test samples of patients suffering
from cardiovascular disease, particularly heart failure. Both hBNP
and human NT-proBNP are frequently used as markers to detect heart
failure and to assess risk thereof in patients. However, the actual
amount of each of the individual forms of BNP (i.e. human proBNP,
human NT-proBNP and human BNP) that circulate is unclear due to the
cross-reactivities of current commercial assays for these various
forms (See, Liang F., et al., J. American College of Cardiology,
49(10):1071-1078 (2007)).
[0010] Additionally, it is known that human proBNP and human
NT-proBNP can be glycosylated (See, Schellenberger, U. et al.,
Archives of Biochemistry and Biophysics, 451:160-166 (2006)), and
these glycosylated forms have been isolated from human samples
(See, Hammerer-Lercher A., et al., Clinical Chemistry,
54(5):858-865 (2008) and Seferian, K. et al., Clinical Chemistry,
54(5):866-873 (2008)). There are seven sites of possible
glycosylation confined to a 36-amino acid region within the N
terminal portion of the peptide (from amino acid 36 through 71).
Antibodies generated to this region may or may not bind to samples
containing analyte human proBNP or NT-proBNP, depending on: 1) the
immunogen used to raise the antibody; and 2) whether or not the
analyte is glycosylated. Optional assays for human proBNP and
NT-proBNP should use antibodies that avoid these regions.
[0011] A number of high-affinity monoclonal antibodies that are
specific for human NT-proBNP and human proBNP are known in the art.
For example, HyTest News, dated June 2005, describes a number of
such high affinity monoclonal antibodies. An example of antibodies
disclosed are shown in the below Table A.
TABLE-US-00001 TABLE A Mab Cat. # Specificity Subclass Epitope
Application 15F11 4NT1 Human NT-proBNP, IgG2b a.a.r. 13-24 EIA,
proBNP Sandwich Immunoassay, WB 24E11 4NT1 Human NT-proBNP, IgG2a
a.a.r. 67-76 EIA, proBNP Sandwich Immunoassay, WB 15C4 4NT1 Human
NT-proBNP, IgG2b a.a.r. 63-71 EIA, proBNP Sandwich Immunoassay, WB
29D12 4NT1 Human NT-proBNP, IgG2a a.a.r. 5-12 EIA, proBNP Sandwich
Immunoassay, WB 13G12 4NT1 Human NT-proBNP, IgG2a a.a.r. 13-20 EIA,
proBNP Sandwich Immunoassay, WB 18H5 4NT1 Human NT-proBNP, IgG1
a.a.r. 13-20 EIA, proBNP Sandwich Immunoassay, WB
[0012] The HyTest News, dated June 2005 describes human NT-proBNP
quantitative sandwich immunoassays and teaches that that monoclonal
antibody pairs (capture-detection) 15F11-24E11, 15C4-29D12,
15C4-13G12 and 15C4-18H5 demonstrate high sensitivity in antigen
recognition (10-15 pg/ml) and good kinetics. The HyTest News is
completely silent on the sensitivity and cross-reactivity of said
monoclonal antibody pairs with any human proBNP that might be in a
test sample. Moreover, human proBNP cross reactivity with human
NT-proBNP kits has been observed in the literature (See,
Luckenbill, K., et al., Clin. Chem., 54:619-621 (2008)).
[0013] In view thereof, there is a need in the art for new assays
for quantifying the amount of human NT-proBNP, particularly assays
having reduced cross-reactivity with other forms of the peptide,
and especially as any clinical significance of variance in their
individual circulating concentrations (e.g., vis-a-vis other forms)
becomes understood. The present disclosure seeks to provide new
assays and methods. The present disclosure also seeks to provide a
kit for use in such assays and methods. The methods and kit can be
used in qualitative or quantitative assays for human NT-proBNP,
including assays carried out to assess the severity of
cardiovascular disease, monitor progression of cardiovascular
disease, or assess risk of progression of cardiovascular disease.
These and other objects and advantages, as well as other additional
features, will become apparent from the detailed description
provided herein.
SUMMARY
[0014] In one embodiment, the present invention relates to an
immunoassay for quantifying the amount of human NT-pro B-type
natriuretic peptide ("human NT-proBNP") in a test sample being
tested for or suspected of containing human NT-proBNP, the
immunoassay having reduced cross-reactivity with any human pro
B-type natriuretic peptide ("human proBNP"). The immunoassay can
comprise the steps of:
[0015] (a) contacting at least one capture antibody that binds to
human NT-proBNP and that has been immobilized onto a solid phase to
produce an immobilized antibody with said test sample to form a
first mixture comprising an at least one capture antibody-human
NT-proBNP complex, wherein the at least one capture antibody
comprises an antibody that binds to an epitope comprising or
consisting of amino acid residues 13-24 of SEQ ID NO:3;
[0016] (b) contacting said first mixture comprising the at least
one capture antibody-human NT-proBNP complex with at least one
detection antibody that binds to human NT-proBNP and that has been
conjugated to a detectable label to form a second mixture
comprising at least one capture antibody-human NT-proBNP-at least
one detection antibody complex, wherein the at least one detection
antibody comprises an antibody that binds to an epitope comprising
or consisting of amino acid residues 63-71 of SEQ ID NO:3 or amino
acid residues 67-76 of SEQ ID NO:3; and
[0017] (c) determining the amount of the at least one capture
antibody-human NT-proBNP-at least one detection antibody complex
formed in step (b) by detecting the detectable label as a measure
of the amount of human NT-proBNP contained in the test sample,
[0018] wherein the at least one capture antibody and the at least
one detection antibody, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP
present in the test sample.
[0019] An example of an antibody that binds to an epitope
comprising or consisting of amino acids residues 13-24 of SEQ ID
NO:3 for use in the above immunoassay is antibody 15F11.
[0020] An example of an antibody that binds to an epitope
comprising or consisting of amino acid residues 63-71 of SEQ ID
NO:3 for use in the above immunoassay is antibody 15C4.
[0021] An example of an antibody that binds to an epitope
comprising or consisting of amino acid residues 67-76 of SEQ ID
NO:3 for use in the above immunoassay is antibody 24E11.
[0022] In another embodiment, the present invention relates to an
immunoassay for quantifying the amount of human NT-proBNP in a test
sample being tested for or suspected of containing human NT-proBNP,
the immunoassay having reduced cross-reactivity with any human
proBNP. The immunoassay can comprise the steps of:
[0023] (a) contacting at least one capture antibody that binds to
human NT-proBNP and that has been immobilized onto a solid phase to
produce an immobilized antibody with said test sample to form a
first mixture comprising an at least one capture antibody-human
NT-proBNP complex, wherein the at least one capture antibody
comprises antibody 15F11;
[0024] (b) contacting said first mixture comprising the at least
one capture antibody-human NT-proBNP complex with at least one
detection antibody that binds to human NT-proBNP and that has been
conjugated to a detectable label to form a second mixture
comprising at least one capture antibody-human NT-proBNP-at least
one detection antibody complex, wherein the at least one detection
antibody comprises antibody 24E11 or 15C4; and
[0025] (c) determining the amount of the at least one capture
antibody-human NT-proBNP-at least one detection antibody complex
formed in step (b) by detecting the detectable label as a measure
of the amount of human NT-proBNP contained in the test sample,
[0026] wherein the at least one capture antibody and the at least
one detection antibody, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP
present in the test sample.
[0027] In the above immunoassay, the at least one detection
antibody can be 24E11.
[0028] Optionally, in the above immunoassay, the at least one
detection antibody can be 15C4.
[0029] In still yet another embodiment, the present invention
relates to an immunoassay for quantifying the amount of human
NT-proBNP in a test sample being tested for or suspected of
containing human NT-proBNP, the immunoassay having reduced
cross-reactivity with any human proBNP present in the test sample.
The immunoassay comprises the steps of:
[0030] (a) contacting said test sample with at least one detection
antibody that binds to human NT-proBNP and that has been conjugated
to a detectable label to form a first mixture comprising an at
least one human NT-proBNP-detection antibody complex, wherein the
at least one detection antibody comprises an antibody that binds to
an epitope comprising or consisting of amino acid residues 63-71 of
SEQ ID NO:3 or amino acid residues 67-76 of SEQ ID NO:3;
[0031] (b) contacting said first mixture comprising said at least
one human NT-proBNP-detection antibody complex with at least one
capture antibody that binds to human NT-proBNP and that has been
immobilized on to a solid phase to produce an immobilized antibody
to form a second mixture comprising an at least one capture
antibody-human NT-proBNP-at least one detection antibody complex,
wherein said at least one capture antibody comprises an antibody
that binds to an epitope comprising or consisting of amino acid
residues 13-24 of SEQ ID NO:3; and
[0032] (c) determining the amount of the at least one capture
antibody-human NT-proBNP-at least one detection antibody complex
formed in step (b) by detecting the detectable label as a measure
of the amount of human NT-proBNP contained in the test sample,
[0033] wherein the at least one capture antibody and the at least
one detection antibody, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP
present in the test sample.
[0034] An example of an antibody that binds to an epitope
comprising or consisting of amino acid residues 13-24 of SEQ ID
NO:3 for use in the above immunoassay is antibody 15F11.
[0035] An example of an antibody that binds to an epitope
comprising or consisting of amino acid residues 63-71 of SEQ ID
NO:3 for use in the above immunoassay is antibody 15C4.
[0036] An example of an antibody that binds to an epitope
comprising or consisting of amino acid residues 67-76 of SEQ ID
NO:3 for use in the above immunoassay is antibody 24E11.
[0037] In still yet another embodiment, the present invention
relates to an immunoassay for quantifying the amount of human
NT-proBNP in a test sample being tested for or suspected of
containing human NT-proBNP, the immunoassay having reduced
cross-reactivity with any human proBNP present in the test sample.
The immunoassay comprises the steps of:
[0038] (a) contacting said test sample with at least one detection
antibody that binds to human NT-proBNP and that has been conjugated
to a detectable label to form a first mixture comprising an at
least one human NT-proBNP-detection antibody complex, wherein the
at least one detection antibody comprises antibody 24E11 or
15C4;
[0039] (b) contacting said first mixture comprising said at least
one human NT-proBNP-detection antibody complex with at least one
capture antibody that binds to human NT-proBNP and that has been
immobilized on to a solid phase to produce an immobilized antibody
to form a second mixture comprising an at least one capture
antibody-human NT-proBNP-at least one detection antibody complex,
wherein said at least one capture antibody comprises antibody
15F11; and
[0040] (c) determining the amount of the at least one capture
antibody-human NT-proBNP-at least one detection antibody complex
formed in step (b) by detecting the detectable label as a measure
of the amount of human NT-proBNP contained in the test sample,
[0041] wherein the at least one capture antibody and the at least
one detection antibody, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP
present in the test sample.
[0042] In the above immunoassay, the at least one detection
antibody can be 24E11.
[0043] Optionally, in the above immunoassay, the at least one
detection antibody can be 15C4.
[0044] In still yet another embodiment, the present invention
relates to an immunoassay for quantifying the amount of human
NT-proBNP in a test sample being tested for or suspected of
containing human NT-proBNP, the immunoassay having reduced
cross-reactivity with any human proBNP present in the test sample.
The immunoassay comprises the steps of:
[0045] (a) contacting a test sample with at least one capture
antibody that binds to human NT-proBNP and that has been
immobilized onto a solid phase to produce an immobilized antibody
and with at least one detection antibody that binds to human
NT-proBNP and that has been conjugated to a detectable label to
form an at least one capture antibody-human NT-proBNP-at least one
detection antibody complex, wherein the at least one capture
antibody comprises an antibody that binds to an epitope comprising
or consisting of amino acid residues 13-24 of SEQ ID NO:3 and the
at least one detection antibody comprises an antibody that binds to
an epitope comprising or consisting of amino acid residues 63-71 of
SEQ ID NO:3 or amino acid residues 67-76 of SEQ ID NO:3; and
[0046] (b) determining the amount of the at least one capture
antibody-human NT-proBNP-- at least one detection antibody complex
formed in step (a) by detecting the detectable label as a measure
of the amount of human NT-proBNP contained in the test sample,
[0047] wherein the at least one capture antibody and the at least
one second antibody conjugated to the detectable label, when used
together, exhibit a cross-reactivity of less than about 1.0% with
any human proBNP present in the test sample.
[0048] An example of an antibody that binds to an epitope
comprising or consisting of amino acid residues 13-24 of SEQ ID
NO:3 for use in the above immunoassay is antibody 15F11.
[0049] An example of an antibody that binds to an epitope
comprising or consisting of amino acids 63-71 of SEQ ID NO:3 for
use in the above immunoassay is antibody 15C4.
[0050] An example of an antibody that binds to an epitope
comprising or consisting of amino acid residues 67-76 of SEQ ID
NO:3 for use in the above immunoassay is antibody 24E11.
[0051] In still yet another embodiment, the present invention
relates to an immunoassay for quantifying the amount of human
NT-proBNP in a test sample being tested for or suspected of
containing human NT-proBNP, the immunoassay having reduced
cross-reactivity with any human proBNP present in the test sample.
The immunoassay comprises the steps of:
[0052] (a) contacting a test sample with at least one capture
antibody that binds to human NT-proBNP and that has been
immobilized onto a solid phase to produce an immobilized antibody
and with at least one detection antibody that binds to human
NT-proBNP and that has been conjugated to a detectable label to
form an at least one capture antibody-human NT-proBNP-at least one
detection antibody complex, wherein the at least one capture
antibody comprises antibody 15F11 and the at least one detection
antibody is antibody 24E11 or 15C4; and
[0053] (b) determining the amount of the at least one capture
antibody-human NT-proBNP-- at least one detection antibody complex
formed in step (a) by detecting the detectable label as a measure
of the amount of human NT-proBNP contained in the test sample,
[0054] wherein the at least one capture antibody and the at least
one second antibody conjugated to the detectable label, when used
together, exhibit a cross-reactivity of less than about 1.0% with
any human proBNP present in the test sample.
[0055] In the above immunoassay, the at least one detection
antibody can be 24E11.
[0056] Optionally, in the above immunoassay, the at least one
detection antibody can be 15C4.
[0057] In still yet another embodiment, the present invention
relates to an immunodiagnostic reagent comprising at least one
capture antibody and at least one detection antibody specific for
human NT-proBNP, and that, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP.
[0058] In the above described immunodiagnostic reagent, the at
least one capture antibody comprises an antibody that binds to an
epitope comprising or consisting of amino acid residues 13-24 of
SEQ ID NO:3 and the at least one detection antibody comprises an
antibody that binds to an epitope comprising or consisting of amino
acid residues 63-71 of SEQ ID NO:3 or amino acid residues 67-76 of
SEQ ID NO:3.
[0059] An example of a capture antibody that binds to an epitope
comprising or consisting of amino acid residues 13-24 of SEQ ID
NO:3 is antibody 15F11.
[0060] An example of a detection antibody that antibody that binds
to an epitope comprising or consisting of amino acid residues 63-71
of SEQ ID NO:3 is antibody 15C4.
[0061] An example of a detection antibody that binds to an epitope
comprising or consisting of amino acid residues 67-76 of SEQ ID
NO:3 is antibody 24E11.
[0062] In still yet another embodiment, the present invention
relates to a kit for the detection of human NT-proBNP in a test
sample. The kit can comprise:
[0063] (a) instructions for conducting an assay of the test sample;
and
[0064] (b) an immunodiagnostic reagent that comprises at least one
capture antibody and at least one detection antibody specific for
human NT-proBNP, and that, when used together, exhibit a
cross-reactivity of less than about 1.0% with any human proBNP.
[0065] In the above kit, the at least one capture antibody
comprises an antibody that binds to an epitope comprising or
consisting of amino acid residues 13-24 of SEQ ID NO:3 and the at
least one detection antibody comprises an antibody that binds to an
epitope comprising or consisting of amino acid residues 63-71 of
SEQ ID NO:3 or amino acid residues 67-76 of SEQ ID NO:3.
[0066] An example of a capture antibody that binds to an epitope
comprising or consisting of amino acid residues 13-24 of SEQ ID
NO:3 is antibody 15F11.
[0067] An example of a detection antibody that antibody that binds
to an epitope comprising or consisting of amino acid residues 63-71
of SEQ ID NO:3 is antibody 15C4.
[0068] An example of a detection antibody that binds to an epitope
comprising or consisting of amino acid residues 67-76 of SEQ ID
NO:3 is antibody 24E11.
DETAILED DESCRIPTION
[0069] The present disclosure relates to immunoassays for
quantifying the amount of human NT-pro B-type natriuretic peptide
("human NT-proBNP") present in a test sample being tested for or
suspected of containing human NT-proBNP. Specifically, the
immunoassays of the present disclosure exhibit reduced
cross-reactivity with any human pro B-type natriuretic peptide
("human proBNP") present in the test sample. In another embodiment,
the present disclosure relates to immunoassays for quantifying the
amount of human NT-proBNP in a test sample wherein the immunoassays
exhibit reduced cross-reactivity with human proBNP. In still yet
another embodiment, the present invention relates to an
immunodiagnostic reagent comprising at least one capture antibody
and at least one detection antibody specific for human NT-proBNP,
and that, when used together, exhibit a cross-reactivity of less
than about 1.0% with any human proBNP present in the test sample.
In still yet another embodiment, the present invention relates to a
kit for performing an immunoassay. Such kits can comprise
instructions for conducting such an immunoassay and the above
described immunodiagnostic reagent.
A. Definitions
[0070] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
[0071] a) Antibody
[0072] As used herein, the terms "antibody" and "antibodies" refer
to monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies (fully or partially humanized),
animal antibodies (in one aspect, a bird (for example, a duck or
goose), in another aspect, a shark or whale, in yet another aspect,
a mammal, including a non-primate (for example, a cow, pig, camel,
llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog,
rat, mouse, etc) and a non-human primate (for example, a monkey,
such as a cynomologous monkey, a chimpanzee, etc)), recombinant
antibodies, chimeric antibodies, single-chain Fvs (scFv), single
chain antibodies, single domain antibodies, Fab fragments, F(ab')
fragments, Fab'' fragments, disulfide-linked Fvs (sdFv), and
anti-idiotypic (anti-Id) antibodies (including, for example,
anti-Id antibodies to antibodies of the present disclosure), and
functionally active epitope-binding fragments of any of the above.
In particular, antibodies include immunoglobulin molecules and
immunologically active fragments of immunoglobulin molecules,
namely, molecules that contain an antigen binding site.
Immunoglobulin molecules can be of any type (for example, IgG, IgE,
IgM, IgD, IgA and IgY), class (for example, IgG.sub.1, IgG.sub.2,
IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2) or subclass.
[0073] b) 15C4
[0074] As used herein, the term "15C4" refers to an IgG2b
monoclonal antibody that is available from HyTest (Turku, Finland)
(Cat. # 4NT1) that binds to amino acid residues 63-71 of SEQ ID
NO:3.
[0075] c) 15F11
[0076] As used herein, the term "15F11" refers to an IgG2b
monoclonal antibody that is available from HyTest (Turku, Finland)
(Cat. # 4NT1) that binds to amino acid residues 13-24 of SEQ ID
NO:3.
[0077] d) 24E11
[0078] As used herein, the term "24E11" refers to an IgG2a
monoclonal antibody that is available from HyTest (Turku, Finland)
(Cat. # 4NT1) that binds to amino acid residues 67-76 of SEQ ID
NO:3.
[0079] e) Epitope
[0080] As used herein, the term "epitope" or "epitopes" refers to
sites or fragments of a polypeptide or protein having antigenic or
immunogenic activity in a subject. An epitope having immunogenic
activity is a site or fragment of a polypeptide or protein that
elicits an antibody response in an animal. An epitope having
antigenic activity is a site or fragment of a polypeptide or
protein to which an antibody immunospecifically binds as determined
by any method well-known to those skilled in the art, for example
by immunoassays.
[0081] f) Human Brain Natriuretic Peptide
[0082] As used herein, the terms "human brain natriuretic peptide",
"human BNP", "hBNP", "hBNP-32", "hBNP peptide", "hBNP polypeptide",
or "B-type natriuretic peptide" used interchangeably herein, refer
to a 32 amino acid molecule having the amino acid sequence shown in
SEQ ID NO:4. The amino acid sequence shown in SEQ ID NO:4 is
represented by amino acid residues 77-108 of the 108 amino acid
sequence of human proBNP (SEQ ID NO:2).
[0083] g) hBNP Fragment
[0084] As used herein, the terms "hBNP fragment" "hBNP-32
fragment", "hBNP peptide fragment" or "human BNP fragment" as used
interchangeably herein refers to a polypeptide that comprises at
least six contiguous amino acid residues of SEQ ID NO:4. In one
aspect, a hBNP fragment or hBNP peptide fragment refers to a
peptide that comprises at least ten contiguous amino acid residues
of SEQ ID NO:4; at least fifteen contiguous amino acid residues of
amino acid residues of SEQ ID NO:4; at least 20 contiguous amino
acid residues of SEQ ID NO:4; at least 25 contiguous amino acid
residues of SEQ ID NO:4, or at least 30 contiguous amino acid
residues of amino acids of SEQ ID NO:4. Examples of hBNP fragments
or hBNP peptide fragments include, but are not limited to, amino
acid sequences containing amino acids residues 1-31, 1-30, 1-29,
1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18,
1-17, 1-16, 1-15, 2-32, 2-31, 2-30, 2-29, 2-28, 2-27, 2-26, 2-25,
2-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14,
2-13, 2-12, 2-11, 2-10, 2-9,2-8, 2-7,3-32, 3-31, 3-30, 3-29, 3-28,
3-27, 3-26, 3-25, 3-24, 3-23, 3-32, 3-21, 3-20, 3-19, 3-18, 3-17,
3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9,3-8, 4-32, 4-31,
4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20,
4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10,
4-9,5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23,
5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12,
5-11, 5-10, 6-32, 6-31, 6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24,
6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13,
6-12, 6-11, 7-32, 7-31, 7-30, 7-29, 7-28, 7-27, 7-26, 7-25, 7-24,
7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13,
7-12, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23,
8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 9-32,
9-31, 9-30, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-22, 9-21,
9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 10-32, 10-31, 10-30,
10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21,
10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 11-32, 11-31, 11-30,
11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21,
11-20, 11-19, 11-18, 11-17 or 11-16 of SEQ ID NO:4.
[0085] h) Immunodiagnostic Reagent
[0086] As used herein, the term "immunodiagnostic reagent" refers
to one or more antibodies that specifically bind to a region (e.g.,
epitope) of human NT-proBNP.
[0087] i) Pre-Pro Peptide Precursor of Human BNP
[0088] As used herein, the term "pre-pro peptide precursor of human
BNP" or "human pre-proBNP" refers to a 134 amino acid molecule
having the amino acid sequence shown in SEQ ID NO:1.
[0089] j) Human Pro B-Type Natriuretic Peptide
[0090] As used herein, the phrase "human pro B-type natriuretic
peptide" or "human proBNP" refers to a 108 amino acid molecule
having the amino acid sequence shown in SEQ ID NO:2. Human proBNP
is derived from human pre-proBNP.
[0091] k) Human N-Terminal-pro B-type Natriuretic Peptide
[0092] As used herein, the phrase "Human N-terminal-pro B-type
natriuretic peptide" or "human NT-proBNP", refers to a 76 amino
acid molecule having the amino acid sequence shown in SEQ ID NO:3.
Human NT-proBNP is derived from human proBNP (SEQ ID NO:2).
[0093] l) Human N-Terminal-pro B-type Natriuretic Peptide
Fragment
[0094] As used herein, the phrases "human N-terminal-pro B-type
natriuretic peptide fragment" or "human NT-proBNP fragment" as used
interchangeably herein refers to a polypeptide that comprises a
fragment of a human NT-proBNP peptide. The fragment of a human
NT-proBNP peptide contains a contiguous or nonlinear epitope of the
human NT-proBNP peptide. The precise boundaries of such an epitope
fragment can be confirmed using ordinary skill in the art.
Specifically, the human NT-proBNP fragment of the present invention
comprises at least two epitopes, specifically a first epitope
comprising or consisting of amino acid residues 13-24 of SEQ ID
NO:3 and a second epitope comprising or consisting of amino acid
residues 63-71 or amino acid residues 67-76 of SEQ ID NO:3.
Examples of human NT-proBNP fragment include, but are not limited
to, amino acid sequences containing amino acid residues 1-75, 1-74,
1-73, 1-72, 1-71, 2-76, 2-75, 2-74, 2-73, 2-72, 2-71, 3-76, 3-75,
3-74, 3-73, 3-72, 3-71, 4-76, 4-75, 4-74, 4-73, 4-72, 4-71, 5-76,
5-75, 5-74, 5-73, 5-72, 5-71, 6-76, 6-75, 6-74, 6-73, 6-72, 6-71,
7-76, 7-75, 7-74, 7-73, 7-72, 7-71, 8-76, 8-75, 8-74, 8-73, 8-72,
8-71, 9-76, 9-75, 9-74, 9-73, 9-72, 9-71, 10-76, 10-75, 10-74,
10-73, 10-72, 10-71, 11-76, 11-75, 11-74, 11-73, 11-72, 11-71,
12-76, 12-75, 12-74, 12-73, 12-72, 12-71, 13-76, 13-75, 13-74,
13-73, 13-72, 13-71 of SEQ ID NO:3.
[0095] m) Subject
[0096] As used herein, the terms "subject" and "patient" are used
interchangeably, although a subject of the disclosure herein need
not necessarily be undergoing or have undergone medical treatment
at the time of the immunoassay. As used herein, the terms "subject"
and "subjects" refer to an animal, in one aspect, a bird (for
example, a duck or goose), in another aspect, a shark or whale, or
in a further aspect, a mammal including, a non-primate (for
example, a cow, pig, camel, llama, horse, goat, rabbit, sheep,
hamsters, guinea pig, cat, dog, rat, and mouse) and a primate (for
example, a monkey, such as a cynomologous monkey, chimpanzee, and a
human). Preferably, the subject is a human.
[0097] n) Test Sample
[0098] As used herein, the term "test sample" refers to a
biological sample derived from tissues, serum, plasma, whole blood,
lymph, CNS fluid, urine or other bodily fluids of a subject that is
being tested for, and/or may be suspected of containing human
NT-proBNP. The test sample can be prepared using routine techniques
known to those skilled in the art.
B. Immunoassays and Immunodiagnostic Reagents
[0099] As mentioned briefly herein, in one embodiment, the present
disclosure relates to immunoassays for the qualitative detection
and/or quantification of human NT-proBNP or human NT-proBNP
fragment in a test sample. The immunoassays described herein
exhibit reduced cross-reactivity with any human proBNP that may be
contained in the test sample.
[0100] The immunoassays of the present disclosure can be conducted
using any format known in the art, such as, but not limited to, a
sandwich format.
[0101] In certain embodiments of the present disclosure, at least
two antibodies are employed to separate and quantify human
NT-proBNP or human NT-proBNP fragment in a test sample. More
specifically, the at least two antibodies bind to certain epitopes
of human NT-proBNP or human NT-proBNP fragment forming an immune
complex which is referred to as a "sandwich". Generally, in the
immunoassays one or more antibodies can be used to capture the
human NT-proBNP or human NT-proBNP fragment in the test sample
(these antibodies are frequently referred to as a "capture"
antibody or "capture" antibodies) and one or more antibodies can be
used to bind a detectable (namely, quantifiable) label to the
sandwich (these antibodies are frequently referred to as the
"detection antibody," "detection antibodies," a "conjugate" or
"conjugates").
[0102] The inventors have discovered that excellent immunoassays,
particularly, sandwich assays, can be performed using certain
combinations of antibodies as the capture and detection antibodies.
More specifically, the at least one capture antibody used in the
present invention is an antibody that binds to an epitope
comprising or consisting of amino acid residues 13-24 of SEQ ID
NO:3. An example of an antibody that binds to an epitope comprising
or consisting of amino acid residues 13-24 of SEQ ID NO:3 is
antibody 15F11. The at least one detection antibody used in the
present invention is an antibody that binds to an epitope
comprising or consisting of amino acid residues 63-71 of SEQ ID
NO:3 or amino acid residues 67-76 of SEQ ID NO:3. An example of an
antibody that binds to an epitope comprising or consisting of amino
acid residues 63-71 of SEQ ID NO:3 is antibody 15C4. An example of
an antibody that binds to an epitope comprising or consisting of
amino acid residues 67-76 of SEQ ID NO:3 is antibody 24E11.
[0103] Immunoassays performed as described herein that employ as at
least one capture antibody that binds to an epitope comprising or
consisting of amino acid residues 13-24 of SEQ ID NO:3 and at least
one detection antibody that binds to an epitope comprising or
consisting of amino acid residues 63-71 of SEQ ID NO:3 or amino
acid residues 67-76 of SEQ ID NO:3, exhibit reduced
cross-reactivity with any human proBNP that may be present in the
test sample. Preferably, the immunoassay exhibits a
cross-reactivity of less than about 1.0% with any human proBNP that
may be present in the test sample. More preferably, the immunoassay
exhibits a cross-reactivity of less than about 0.9%, less than
about 0.8%, less than about 0.7%, less than about 0.6%, less than
about 0.5%, less than about 0.4%, less than about 0.3%, less than
about 0.2%, less than about 0.1% or less than about 0.001% with any
human proBNP that may be present in the test sample.
[0104] In another embodiment of the disclosure, immunoassays
performed as described herein that employ as at least one capture
antibody that binds to an epitope comprising or consisting of amino
acid residues 13-24 of SEQ ID NO:3 and at least one detection
antibody that binds to an epitope comprising or consisting of amino
acid residues 63-71 of SEQ ID NO:3 or amino acid residues 67-76 of
SEQ ID NO:3, exhibit a reduced cross-reactivity with an human
proBNP that may be present in the test sample. Preferably, the
immunoassay exhibits a cross-reactivity of less than about 1.0%
with any human proBNP that may be present in the test sample. More
preferably, the immunoassay exhibits a cross-reactivity of less
than about 0.9%, less than about 0.8%, less than about 0.7%, less
than about 0.6%, less than about 0.5%, less than about 0.4%, less
than about 0.3%, less than about 0.2%, less than about 0.1% or less
than about 0.001% with any human proBNP that may be present in the
test sample.
[0105] The test sample being tested for (e.g., suspected of
containing) human NT-proBNP or a human NT-proBNP fragment can be
contacted with at least one capture antibody (or antibodies) and at
least one detection antibody (or antibodies) either simultaneously
or sequentially and in any order. For example, the test sample can
be first contacted with at least one capture antibody and then
(sequentially) with at least one detection antibody. Alternatively,
the test sample can be first contacted with at least one detection
antibody and then (sequentially) with at least one capture
antibody. In yet another alternative, the test sample can be
contacted simultaneously with a capture antibody and a detection
antibody.
[0106] In the sandwich assay format, a test sample suspected of
containing human NT-proBNP or human NT-proBNP fragment is first
brought into contact with the at least one first capture antibody
that binds to an epitope comprising or consisting of amino acid
residues 13-24 of SEQ ID NO:3 under conditions which allow the
formation of a first antibody-human NT-proBNP complex. If more than
one capture antibody is used, a first multiple capture
antibody-human NT-proBNP complex is formed. In a sandwich assay,
the antibodies, preferably, the at least one capture antibody, are
used in molar excess amounts of the maximum amount of human
NT-proBNP or human NT-proBNP fragment expected in the test sample.
For example, from about 5 .mu.g/mL to about 1 mg/mL of antibody per
mL of buffer (e.g., microparticle coating buffer) can be used.
[0107] Optionally, prior to contacting the test sample with the at
least one capture antibody (e.g., the first capture antibody), the
at least one capture antibody can be bound to a solid support which
facilitates the separation of the first antibody-human NT-proBNP
complex from the test sample. Any solid support known in the art
can be used, including but not limited to, solid supports made out
of polymeric materials in the forms of wells, tubes or beads. The
antibody (or antibodies) can be bound to the solid support by
adsorption, by covalent bonding using a chemical coupling agent or
by other means known in the art, provided that such binding does
not interfere with the ability of the antibody to bind human
NT-proBNP or human NT-proBNP fragment. Alternatively, the antibody
(or antibodies) can be bound with microparticles that have
previously been coated with streptavidin (for example, using
Power-Bind.TM.-SA-MP streptavidin coated microparticles, available
from Seradyn, Indianapolis, Ind.). Alternatively, the antibody (or
antibodies) can be bound using microparticles that have been
previously coated with anti-species specific monoclonal antibodies.
Moreover, if necessary, the solid support can be derivatized to
allow reactivity with various functional groups on the antibody.
Such derivatization requires the use of certain coupling agents
such as, but not limited to, maleic anhydride, N-hydroxysuccinimide
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
[0108] After the test sample being tested for and/or suspected of
containing human NT-proBNP or an human NT-proBNP fragment is
brought into contact with the at least one capture antibody (e.g.,
the first capture antibody), the mixture is incubated in order to
allow for the formation of a first antibody (or multiple
antibody)-human NT-proBNP complex. The incubation can be carried
out at a pH of from about 4.5 to about 10.0, at a temperature of
from about 2.degree. C. to about 45.degree. C., and for a period
from at least about one (1) minute to about eighteen (18) hours,
preferably from about 1 to 20 minutes, most preferably from about
2-4 minutes. The immunoassay described herein can be conducted in
one step (meaning the test sample, at least one capture antibody
and at least one detection antibody are all added sequentially or
simultaneously to a reaction vessel) or in more than one step, such
as two steps, three steps, etc.
[0109] After formation of the (first/multiple) capture
antibody-human NT-proBNP complex, the complex is then contacted
with at least one detection antibody (under conditions which allow
for the formation of a (first/multiple) capture antibody-human
NT-proBNP-second antibody detection complex). The at least one
detection antibody can be the second, third, fourth, etc.
antibodies used in the immunoassay. If the capture antibody-human
NT-proBNP complex is contacted with more than one detection
antibody, then a (first/multiple) capture antibody-human
NT-proBNP-(multiple) detection antibody complex is formed. As with
the capture antibody (e.g., the first capture antibody), when the
at least second (and subsequent) detection antibody is brought into
contact with the capture antibody-human NT-proBNP complex, a period
of incubation under conditions similar to those described above is
required for the formation of the (first/multiple) capture
antibody-human NT-proBNP-(second/multiple) detection antibody
complex. Preferably, at least one detection antibody contains a
detectable label. The detectable label can be bound to the at least
one detection antibody (e.g., the second detection antibody) prior
to, simultaneously with or after the formation of the
(first/multiple) capture antibody-human NT-proBNP-(second/multiple)
detection antibody complex. Any detectable label known in the art
can be used. For example, the detectable label can be a radioactive
label, such as, .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P,
.sup.33P, an enzymatic label, such as horseradish peroxidase,
alkaline phosphatase, glucose 6-phosphate dehydrogenase, etc., a
chemiluminescent label, such as, acridinium esters, luminol,
isoluminol, thioesters, sulfonamides, phenanthridinium esters, etc.
a fluorescence label, such as, fluorescein (5-fluorescein,
6-carboxyfluorescein, 3'6-carboxyfluorescein,
5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,
6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.),
rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (zinc
sulfide-capped cadmium selenide), a thermometric label or an
immuno-polymerase chain reaction label. An introduction to labels,
labeling procedures and detection of labels is found in Polak and
Van Noorden, Introduction to Immunocytochemistry, 2.sup.nd ed.,
Springer Verlag, N.Y. (1997) and in Haugland, Handbook of
Fluorescent Probes and Research Chemicals(1996), which is a
combined handbook and catalogue published by Molecular Probes,
Inc., Eugene, Oreg. In addition, more than one label can be used.
For example, double conjugates can be used, each of which contain
different labels. For example, one conjugate antibody can contain
biotin and the second conjugate can be an anti-biotin antibody
labeled with acridinium. Other variations would be easily
recognized by one of ordinary skill in the art.
[0110] The detectable label can be bound to the antibodies either
directly or through a coupling agent. An example of a coupling
agent that can be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, hydrochloride) that is commercially available from
Sigma-Aldrich, St. Louis, Mo. Other coupling agents that can be
used are known in the art. Methods for binding a detectable label
to an antibody are known in the art. Additionally, many detectable
labels can be purchased or synthesized that already contain end
groups that facilitate the coupling of the detectable label to the
antibody, such as,
N10-(3-sulfopropyl)-N-(3-carboxypropyl)-acridinium-9-carboxamide
active esters, otherwise known as CPSP-Acridinium Ester or
N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide
active ester, otherwise known as SPSP-Acridinium Ester.
[0111] The (first/multiple) capture antibody-human
NT-proBNP-(second/multiple) detection antibody complex can be, but
does not have to be, separated from the remainder of the test
sample prior to quantification of the label. For example, if the at
least one capture antibody (e.g., the first capture antibody) is
bound to a solid support, such as a well or a bead, separation can
be accomplished by removing the fluid (of the test sample) from
contact with the solid support. Alternatively, if the at least
first capture antibody is bound to a solid support it can be
simultaneously contacted with the human NT-proBNP-containing sample
and the at least one second detection antibody to form a first
(multiple) antibody-human NT-proBNP-second (multiple) antibody
complex, followed by removal of the fluid (test sample) from
contact with the solid support. If the at least one first capture
antibody is not bound to a solid support, then the (first/multiple)
capture antibody-human NT-proBNP-(second/multiple) detection
antibody complex does not have to be removed from the test sample
for quantification of the amount of the label.
[0112] After formation of the labeled capture antibody-human
NT-proBNP-detection antibody complex (e.g., the first capture
antibody-human NT-proBNP-second detection antibody complex), the
amount of label in the complex is quantified using techniques known
in the art. For example, if an enzymatic label is used, the labeled
complex is reacted with a substrate for the label that gives a
quantifiable reaction such as the development of color. If the
label is a radioactive label, the label is quantified using a
scintillation counter. If the label is a fluorescent label, the
label is quantified by stimulating the label with a light of one
color (which is known as the "excitation wavelength") and detecting
another color (which is known as the "emission wavelength") that is
emitted by the label in response to the stimulation. If the label
is a chemiluminescent label, the label is quantified detecting the
light emitted either visually or by using luminometers, x-ray film,
high speed photographic film, a CCD camera, etc. Once the amount of
the label in the complex has been quantified, the concentration of
human NT-proBNP or human NT-proBNP fragment in the test sample is
determined by use of a standard curve that has been generated using
serial dilutions of human NT-proBNP or human NT-proBNP fragment of
known concentration. Other than using serial dilutions of human
NT-proBNP or human NT-proBNP fragment, the standard curve can be
generated gravimetrically, by mass spectroscopy and by other
techniques known in the art.
[0113] In another embodiment, the present disclosure relates to
immunodiagnostic reagents. Specifically, the at least one capture
antibody (namely, an antibody that binds to an epitope comprising
or consisting of amino acid residues 13-24 of SEQ ID NO:3) and at
least one detection antibody (namely, an antibody that binds to an
epitope comprising or consisting of amino acid residues 63-71 of
SEQ ID NO:3 or amino acid residues 67-76 of SEQ ID NO:3) described
herein can be used individually or in combination, as one or more
immunodiagnostic reagents in one or more immunoassays, such as
those described above. When the at least one capture antibody and
at least one detection antibody described herein are used together
in an immunoassay (such as those described previously herein), the
immunoassay exhibits a cross-reactivity of less than about 1.0%
with any human proBNP in a test sample. More specifically, the
immunoassay exhibits less than about 0.9%, less than about 0.8%,
less than about 0.7%, less than about 0.6%, less than about 0.5%,
less than about 0.4%, less than about 0.3%, less than about 0.2%,
less than about 0.1% or less than about 0.001% with any human
proBNP that may be present in the test sample.
C. Adaptations of the Methods
[0114] The disclosure herein also can be adapted for use in a
variety of automated and semi-automated systems (including those
wherein the solid phase comprises a microparticle), as described,
e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as, e.g.,
commercially marketed by Abbott Laboratories (Abbott Park, Ill.)
including but not limited to Abbott's ARCHITECT.RTM., AxSYM.RTM.,
IMx.RTM., PRISM.RTM., and Quantum.TM. II instruments, as well as
other platforms. Moreover, the disclosure optionally is adaptable
for the Abbott Laboratories commercial Point of Care (i-STAT.TM.)
electrochemical immunoassay system for performing sandwich
immunoassays. Immunosensors, and their methods of manufacture and
operation in single-use test devices are described, for example in,
U.S. Pat. No. 5,063,081, U.S. Patent Application 2003/0170881, U.S.
Patent Application 2004/0018577, U.S. Patent Application
2005/0054078, and U.S. Patent Application 2006/0160164, which are
incorporated in their entireties by reference for their teachings
regarding same.
D. Exemplary Kits
[0115] The present disclosure herein also can be adapted for use in
a variety of kits for use on automated and semi-automated systems
and platforms, e.g., commercially marketed by Abbott Laboratories
(Abbott Park, Ill.) including, but not limited to, Abbott
Laboratories' ARCHITECT.RTM., AxSYM.RTM., IMx.RTM., PRISM.RTM., and
Quantum.TM. II instruments, Abbott Laboratories' commercial Point
of Care (i-STAT.TM.) electrochemical immunoassay system for
performing sandwich immunoassays, as well as other platforms.
[0116] Such kits can comprise one or more of the immunodiagnostic
reagents (e.g., the capture and detection antibodies) described
herein. More specifically, if the kit is a kit for performing an
immunoassay, the kit can optionally contain (1) at least one
capture and detection antibody that bind to human NT-proBNP or
human NT-proBNP fragment and together exhibit reduced
cross-reactivity with human proBNP; and (2) one or more
instructions for performing the immunoassay. The immunodiagnostic
reagents of the present disclosure can be included in such a test
kit as a capture antibody, as a detection antibody or both as a
capture antibody and a detection antibody. For example, antibody
15F11 can be included in the kit as capture antibody and antibody
15C4 can be included in the kit as a detection antibody.
Alternatively, antibody 15F11 can be included in the kit as a
capture antibody and antibody 24E11 can be included in the kit as a
detection antibody. Optionally, the kit can also contain at least
one calibrator or control. Any calibrator or control can be
included in the kit. Preferably, however, the calibrator or control
is human NT-proBNP or human NT-proBNP fragment, especially SEQ ID
NO: 3 described previously herein. Accordingly, the kits can
comprise at least one calibrator, or at least one control, or a
combination of at least one calibrator and at least one
control.
[0117] Optionally the kits also can include quality control
reagents (e.g., sensitivity panels, calibrators, and positive
controls). Preparation of quality control reagents is well known in
the art, and is described, e.g., on a variety of immunodiagnostic
product insert sheets. Human NT-proBNP sensitivity panel members
optionally can be prepared in varying amounts containing, e.g.,
known quantities of human NT-proBNP antigen ranging from "low" to
"high", e.g., by spiking known quantities of the human NT-proBNP
antigen into an appropriate assay buffer (e.g., a phosphate
buffer). These sensitivity panel members optionally are used to
establish assay performance characteristics, and further optionally
are useful indicators of the integrity of the immunoassay kit
reagents, and the standardization of assays. The human NT-proBNP
antigen also can be employed as calibrators.
[0118] The antibodies provided in the kit can incorporate a
detectable label, such as a fluorophore, radioactive moiety,
enzyme, biotin/avidin label, chromophore, chemiluminescent label,
or the like, or the kit may include reagents for labeling the
antibodies or reagents for detecting the antibodies (e.g.,
detection antibodies) and/or for labeling the antigens or reagents
for detecting the antigen. The antibodies, calibrators and/or
controls can be provided in separate containers or pre-dispensed
into an appropriate assay format, for example, into microtiter
plates.
[0119] In yet another embodiment, the kit can comprise, either
alone, with instructions, or with other aspects of the kit and kit
components, an immunodiagnostic agent that comprises one or more
antibodies selected from the group consisting of 15F11, 15C4 and
24E11.
[0120] The kits can optionally include other reagents required to
conduct a diagnostic assay or facilitate quality control
evaluations, such as buffers, salts, enzymes, enzyme co-factors,
substrates, detection reagents, and the like. Other components,
such as buffers and solutions for the isolation and/or treatment of
a test sample (e.g., pretreatment reagents), may also be included
in the kit. The kit may additionally include one or more other
controls. One or more of the components of the kit may be
lyophilized and the kit may further comprise reagents suitable for
the reconstitution of the lyophilized components.
[0121] The various components of the kit optionally are provided in
suitable containers. As indicated above, one or more of the
containers may be a microtiter plate. The kit further can include
containers for holding or storing a sample (e.g., a container or
cartridge for a blood or urine sample). Where appropriate, the kit
may also optionally contain reaction vessels, mixing vessels and
other components that facilitate the preparation of reagents or the
test sample. The kit may also include one or more instruments for
assisting with obtaining a test sample, such as a syringe, pipette,
forceps, measured spoon, or the like.
[0122] The kit further can optionally include instructions for use,
which may be provided in paper form or in computer-readable form,
such as a disc, CD, DVD or the like.
[0123] Now by way of example, and not of limitation, examples of
the present disclosure shall now be given.
Example 1
Elimination of Detection of Human proBNP During Immunoassay for
Human NT-proBNP
[0124] Materials, Methods and Results In this example, an automated
ARCHITECT.RTM. System (Abbott Laboratories, Abbott Park, Ill.) was
used to perform an immunoassay that would quantitate human
NT-proBNP in specimens that contained a range of concentrations of
human NT proBNP (HyTest, Turku, Finland; Catalog No. 8NT1) in BNP
Calibrator Diluent which contains 10 mM NaOAc, 10 mM DTPA, 2% BSA,
0.1% ProClin 300, 0.1% NaN.sub.3, pH 5.6 (Abbott Laboratories,
Abbott Park, Ill.).
[0125] Dilutions of human NT proBNP (HyTest, Turku, Finland;
Catalog No. 8NT1) containing human NT proBNP, (0.0 .mu.M-577.4
.mu.M) were tested. Testing was performed in reaction vessels
(Abbott Laboratories, Abbott Park, Ill.) that are used for
individual tests in the automated Abbott ARCHITECT.RTM. System. All
the described steps took place in the ARCHITECT.RTM.
instrument.
[0126] Dilutions of human proBNP (HyTest, Turku, Finland; Catalog
No. 8PRO8) (0.0 .mu.M-577.4 .mu.M) and glycosylated human proBNP
(HyTest, Turku, Finland; Catalog No. 8G0B2) (0.0 .mu.M-577.4 .mu.M)
were simultaneously tested to determine the extent of cross
reactivity. Testing was performed in reaction vessels (Abbott
Laboratories, Abbott Park, Ill.) that are used for individual tests
in the automated Abbott ARCHITECT.RTM. System. All the described
steps took place in the ARCHITECT.RTM. instrument.
[0127] Specimens containing human NT-proBNP (HyTest, Turku,
Finland; Catalog No. 8NT1), human proBNP (HyTest, Turku, Finland;
Catalog No. 8PRO8), and glycosylated human proBNP ((HyTest, Turku,
Finland; Catalog No. 8G0B2) were tested using a traditional
two-step immunoassay format, as described below.
[0128] The two-step assay format occurs in a single reaction
vessel. The two-step assay format comprises the steps of adding the
sample, adding antibody coated magnetic microparticles, binding
analyte, washing magnetic microparticle-analyte complexes, adding
labeled conjugate antibody, binding of labeled conjugate to
magnetic microparticle-analyte complexes, washing the resulting
magnetic microparticle-analyte-labeled conjugate antibody
complexes, and reading signal generated by the complexed label
remaining in the reaction vessel.
[0129] Each sample dilution was dispensed in the amount of 100
.mu.L into the individual reaction vessels. At the same time, 0.10%
EDAC-coated magnetic microparticles (50 .mu.L) (Polymer Science,
Monticello, Ind.) coated with anti-human NT proBNP/anti-human
proBNP monoclonal antibodies 15F11 (HyTest, Turku, Finland; Catalog
No. 4TN1) or 15C4 (HyTest, Turku, Finland; Catalog No. 4TN1) were
dispensed in the amount of 50 .mu.L into the same reaction vessel.
The reaction vessel was then vortexed to mix the sample and
magnetic microparticles. Each reaction mixture was incubated for 18
minutes at 37.degree. C.
[0130] During this incubation, any human NT-proBNP, human proBNP,
or glycosylated human proBNP in the sample was capable of being
captured by the anti-human NT-proBNP/anti-human proBNP monoclonal
antibody coated onto the magnetic microparticles.
[0131] Upon completion of the 4 minute incubation, the magnetic
microparticle human NT-proBNP/human proBNP antibody complexes were
magnetically captured, and immobilized into a pellet on the side of
the reaction vessel. The immobilized magnetic microparticle-human
proBNP/human proBNP complex pellet was then washed by alternately
aspirating the liquid from the vessel, and then adding assay kit
wash buffer (ARCHITECT.RTM. wash buffer, available from Abbott
Laboratories, Abbott Park, Ill.) into the reaction vessel (1 mL
wash buffer, repeated 4 times). This process removes any unbound
human NT-proBNP, human proBNP, and glycosylated human proBNP from
reaction mixture. The magnetically captured microparticle-human
NT-proBNP/human proBNP antibody complexes formed during the 4
minute incubation remain in the reaction vessel.
[0132] During a second incubation of 4 minutes, anti-human
NT-proBNP/anti-human proBNP labeled monoclonal antibody 7B5
(HyTest, Turku, Finland; Catalog No. 4TN1) or 15C4 (HyTest, Turku,
Finland; Catalog No. 4TN1) or 15F11(HyTest, Turku, Finland; Catalog
No. 4TN1) or 16F3 (HyTest, Turku, Finland; Catalog No. 4TN1) or
13G12 (HyTest, Turku, Finland; Catalog No. 4TN1) or 18H5 (HyTest,
Turku, Finland; Catalog No. 4TN1) or 24E11 (HyTest, Turku, Finland;
Catalog No. 4TN1) or 29D12 (HyTest, Turku, Finland; Catalog No.
4TN1) were each dispensed in the amount of 50 .mu.L into individual
reaction vessels containing only the 15F11 (HyTest, Turku, Finland;
Catalog No. 4TN1) coated magnetic microparticles-human
NT-proBNP/human proBNP complexes. This reaction mixture was then
vortexed to disperse the microparticle pellet.
[0133] Also, during a second incubation of 4 minutes, anti-human
NT-proBNP/human anti-proBNP labeled monoclonal antibody 7B5
(HyTest, Turku, Finland; Catalog No. 4TN1) or 15C4 (HyTest, Turku,
Finland; Catalog No. 4TN1) or 15F11(HyTest, Turku, Finland; Catalog
No. 4TN1) or 16F3 (HyTest, Turku, Finland; Catalog No. 4TN1) or
13G12 (HyTest, Turku, Finland; Catalog No. 4TN1) or 18H5 (HyTest,
Turku, Finland; Catalog No. 4TN1) or 24E11 (HyTest, Turku, Finland;
Catalog No. 4TN1) or 29D12 (HyTest, Turku, Finland; Catalog No.
4TN1) were each dispensed in the amount of 50 .mu.L into individual
reactions vessel containing only the 15C4 (HyTest, Turku, Finland;
Catalog No. 4TN1) coated magnetic microparticle-human
NT-proBNP/human proBNP complexes. This reaction mixture was then
vortexed to disperse the microparticle pellet.
[0134] The magnetic microparticle-human NT-proBNP/human proBNP
complexes were incubated with the labeled antibodies in buffer for
4 minutes at 37.degree. C. During this incubation, the anti-human
NT-proBNP/human anti-proBNP acridinium labeled antibodies also bind
to the human NT-proBNP or human proBNP that was captured by the
magnetic microparticles. This formed a microparticle-human NT
proBNP or human proBNP-labeled antibody complex.
[0135] Upon completion of the 4 minute incubation, the magnetic
microparticle-human NT-proBNP/human proBNP-labeled antibody
complexes were again magnetically captured into a pellet. The
recaptured pellet was then repeatedly washed with buffer (1 mL),
repeated 4 times. The magnetically captured microparticle-human NT
proBNP or human proBNP-labeled antibody complex pellet was then
released.
[0136] The acridinium label (Abbott Laboratories, Abbott Park,
Ill.) was then triggered to emit light. This was accomplished by
adding a low pH (pH 1) buffer containing H.sub.2O.sub.2 (1.32%)
(List 6E23-65, Abbott Laboratories, Abbott Park, Ill.) was
dispensed (100 .mu.L) to the microparticle complexes and vortexing.
This step released the labeled anti-human NT-proBNP/anti-human
proBNP acridinium (Abbott Laboratories, Abbott Park, Ill.)
monoclonal antibody (Abbott Laboratories, Abbott Park, Ill.) that
had been bound to human NT-proBNP or human proBNP captured by the
microparticles.
[0137] The magnetic microparticles were then magnetically captured
leaving the released acridinium labeled NT-proBNP or proBNP
antibodies in the reaction mixture solution. This was followed by
addition (300 .mu.L) of a pH 13 buffer (List 6C55-60, Abbott
Laboratories, Abbott Park, Ill.) which "triggers" light, relative
light units (RLU), production from the acridinium released into the
solution. The amount of light that was generated was used to
determine the quantity of human NT-human proBNP, human proBNP, and
glycosylated human proBNP detected in the sample (See, Table
1).
TABLE-US-00002 TABLE 1 Analyte rlus Microparticle Conjugate Human
Human Human Mab Mab pM NT-proBNP gly. proBNP proBNP 15F11 7B5 0.0
615 615 615 43.3 615 528 589 86.6 549 505 529 288.7 593 525 556
577.4 534 600 612 15F11 15C4 0.0 1345 1345 1345 43.3 21153 1263
1490 86.6 57226 2009 1544 288.7 312467 1577 2190 577.4 661509 1452
3358 15F11 15F11 0.0 912 912 912 43.3 904 899 857 86.6 929 886 963
288.7 977 925 949 577.4 1011 953 897 15F11 16F3 0.0 640 640 640
43.3 630 586 624 86.6 592 587 621 288.7 696 643 720 577.4 631 635
914 15F11 13G12 0.0 841 841 841 43.3 777 798 800 86.6 761 767 802
288.7 820 831 920 577.4 805 778 1076 15F11 18H5 0.0 626 626 626
43.3 621 618 628 86.6 636 737 664 288.7 708 663 949 577.4 664 754
1445 15F11 24E11 0.0 886 886 886 43.3 7381 863 904 86.6 20757 918
938 288.7 124174 906 1091 577.4 356904 892 1244 15F11 29D12 0.0 912
912 912 43.3 1986 2325 1569 86.6 3535 4474 2206 288.7 14573 16406
6145 577.4 33877 38714 11395
TABLE-US-00003 TABLE 2 Analyte rlus Micropartic Conjugate Human
Human Human Mab Mab pM NT-proBNP gly. proBNP proBNP 15C4 7B5 0.0
590 590 590 43.3 750 606 686 86.6 719 581 641 288.7 1178 672 607
577.4 2182 800 726 15C4 15C4 0.0 1941 1941 1941 43.3 1975 1805 2175
86.6 1995 1877 1824 288.7 2057 1862 1884 577.4 2078 1793 1955 15C4
15F11 0.0 847 847 847 43.3 883 1011 851 86.6 998 835 1035 288.7
1135 930 945 577.4 1372 859 917 15C4 16F3 0.0 1036 1036 1036 43.3
11382 691 1987 86.6 26463 861 4059 288.7 140366 827 13737 577.4
319526 1092 34681 15C4 13G12 0.0 892 892 892 43.3 10281 905 2217
86.6 25550 912 4076 288.7 131068 967 16707 577.4 308579 1242 37162
15C4 18H5 0.0 704 704 704 43.3 18028 756 3384 86.6 39142 783 6590
288.7 161543 1228 ND 577.4 336945 1582 46534 15C4 24E11 0.0 821 821
821 43.3 786 781 718 86.6 699 751 747 288.7 814 725 834 577.4 850
770 735 15C4 29D12 0.0 892 892 892 43.3 8867 877 2167 86.6 19329
1017 3834 288.7 82751 1222 12082 577.4 179728 1416 25910
TABLE-US-00004 TABLE 3 % Cross-reactivity Magnetic Acridinium Human
NT-proBNP Micropartic Conjugate Human Human Mab Mab pM gly. proBNP
proBNP 15C4 16F3 43.3 -3.3 9.2 86.6 -0.7 11.9 288.7 -0.2 9.1 577.4
0.0 10.6 15C4 13G12 43.3 0.1 14.1 86.6 0.1 12.9 288.7 0.1 12.1
577.4 0.1 11.8 15C4 18H5 43.3 0.3 15.5 86.6 0.2 15.3 288.7 0.3 nd
577.4 0.3 13.6 15C4 29D12 43.3 -0.2 16.0 86.6 0.7 16.0 288.7 0.4
13.7 577.4 0.3 14.0 15F11 15C4 43.3 -0.4 0.7 86.6 1.2 0.4 288.7 0.1
0.3 577.4 0.0 0.3 15F11 24E11 43.3 -0.4 0.3 86.6 0.2 0.3 288.7 0.0
0.2 577.4 0.0 0.1 15F11 29D12 43.3 131.6 61.2 86.6 135.8 49.3 288.7
113.4 38.3 577.4 114.7 31.8 *Note, in this Table 3, some
combinations were omitted because of a lack of significant
reactivity.
[0138] Discussion of the Results
[0139] A two step sandwich immunoassay was used to quantitate human
NT-proBNP, human glycosylated proBNP and human proBNP with HyTest
anti-human NT-proBNP/anti human proBNP monoclonal antibodies.
[0140] Two anti-human NT-proBNP/anti-human proBNP monoclonal
antibodies were separately coated onto magnetic microparticles and
used for human NT-proBNP, human glycosylated proBNP, and human
proBNP immunoassay capture. Eight labeled anti-human
NT-proBNP/anti-human proBNP monoclonal antibodies were used for
human NT-proBNP, human glycosylated proBNP and human proBNP
immunoassay detection. A total of sixteen combinations of HyTest
anti-human NT-proBNP/anti-human proBNP monoclonal antibodies were
tested (See, Table 4).
TABLE-US-00005 TABLE 4 Magnetic Labeled Microparticles Epitope
Conjugate Epitope Coating Mab a.a. Mab a.a. 15C4 63-71 7B5 13-24
15C4 63-71 15C4 63-71 15C4 63-71 15F11 13-24 15C4 63-71 16F3 13-20
15C4 63-71 13G12 13-20 15C4 63-71 18H5 13-20 15C4 63-71 24E11 67-76
15C4 63-71 29D12 5-12 15F11 13-24 7B5 13-24 15F11 13-24 15C4 63-71
15F11 13-24 15F11 13-24 15F11 13-24 16F3 13-20 15F11 13-24 13G12
13-20 15F11 13-24 18H5 13-20 15F11 13-24 24E11 67-76 15F11 13-24
29D12 5-12
[0141] Seven of the tested combinations detected human NT-proBNP
(See, Table 5). Four of these combinations (15C4 anti-human
NT-proBNP/human anti-proBNP monoclonal antibody coated magnetic
microparticles used with labeled antibodies 16F3, 13G12, 18H5, and
29D12 (anti-human NT-proBNP/human anti-proBNP monoclonal
antibodies)) also cross-reacted between 9% and 15% with equimolar
concentrations of human proBNP (See, Table 3). This result was not
unexpected since human proBNP shares all the epitopes found on
human NT-proBNP.
[0142] 15F11 anti-human NT-proBNP/human anti-proBNP monoclonal
antibody coated magnetic microparticles tested with 29D 12 labeled,
anti-human NT-proBNP/human anti-proBNP monoclonal antibody, reacted
relatively weakly (33877 rlus, 577 pM) with human NT-proBNP
compared to (38714 rlus, 577 pM) with human glycosylated proBNP,
and (11395 rlus, 577 pM) with human pro-BNP (See, Table 1). This
result was not unexpected considering human NT-proBNP also shares
epitopes with human glycosylated proBNP and human proBNP.
[0143] Two anti-human NT-proBNP/anti-human proBNP monoclonal
combinations using 15F11 anti-human NT-proBNP/human anti-proBNP
monoclonal antibody coated onto magnetic microparticles, with
either 15C4 or 24E11 anti-human NT-proBNP/human anti-proBNP
monoclonal labeled antibodies, readily detected human NT-proBNP but
cross reacted with human proBNP less than about 1%. (See, Table 3).
This finding was surprising and unexpected. Specifically,
considering that human NT-proBNP shares the same epitopes with
human glycosylated proBNP and human proBNP, one skilled in the art
would expect that human proBNP would also be detected.
[0144] The anti-human NT/proBNP monoclonal antibodies combinations
which readily detect human NT-proBNP, but do not cross-react with
human glycosylated proBNP (e.g., less than about <1%) or human
proBNP (e.g., less than about <1%), are based on human NT-proBNP
amino acid residues 13-34 (15F11) magnetic microparticle capture
and human NT proBNP amino acids residues 63-71 (15C4) or amino acid
residues 67-76 (24E11) for detection.
[0145] The anti-human NT/proBNP monoclonal antibodies combinations
that used a reverse amino acid capture (13-20), and detection
(63-71), resulted in significant cross-reactivity (-10%) with human
proBNP.
TABLE-US-00006 TABLE 5 Microparticle Epitope Conjugate Epitope
Human Human Human Mab Amino A. Mab Amino A. NT-proBNP gly. proBNP
proBNP 15C4 63-71 16F3 13-20 + - + 15C4 63-71 13G12 13-20 + - +
15C4 63-71 18H5 13-20 + - + 15C4 63-71 29D12 5-12 + - + 15F11 13-24
15C4 63-71 + - - 15F11 13-24 24E11 67-76 + - - 15F11 13-24 29D12
5-12 + + +
[0146] One skilled in the art would readily appreciate that the
present disclosure is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The molecular complexes and the methods, procedures,
treatments, molecules, specific compounds described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
It will be readily apparent to one skilled in the art that varying
substitutions and modifications may be made to the invention
disclosed herein without departing from the scope and spirit of the
invention.
[0147] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0148] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising,"
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present disclosure has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
Sequence CWU 1
1
41134PRTHomo sapiens 1Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu
Leu Leu Leu Leu Phe1 5 10 15Leu His Leu Ala Phe Leu Gly Gly Arg Ser
His Pro Leu Gly Ser Pro 20 25 30Gly Ser Ala Ser Asp Leu Glu Thr Ser
Gly Leu Gln Glu Gln Arg Asn 35 40 45His Leu Gln Gly Lys Leu Ser Glu
Leu Gln Val Glu Gln Thr Ser Leu 50 55 60Glu Pro Leu Gln Glu Ser Pro
Arg Pro Thr Gly Val Trp Lys Ser Arg65 70 75 80Glu Val Ala Thr Glu
Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr 85 90 95Thr Leu Arg Ala
Pro Arg Ser Pro Lys Met Val Gln Gly Ser Gly Cys 100 105 110Phe Gly
Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys 115 120
125Lys Val Leu Arg Arg His 1302108PRTHomo sapiens 2His Pro Leu Gly
Ser Pro Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly1 5 10 15Leu Gln Glu
Gln Arg Asn His Leu Gln Gly Lys Leu Ser Glu Leu Gln 20 25 30Val Glu
Gln Thr Ser Leu Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr 35 40 45Gly
Val Trp Lys Ser Arg Glu Val Ala Thr Glu Gly Ile Arg Gly His 50 55
60Arg Lys Met Val Leu Tyr Thr Leu Arg Ala Pro Arg Ser Pro Lys Met65
70 75 80Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser
Ser 85 90 95Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 100
105376PRTHomo sapiens 3Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu
Leu Leu Leu Leu Phe1 5 10 15Leu His Leu Ala Phe Leu Gly Gly Arg Ser
His Pro Leu Gly Ser Pro 20 25 30Gly Ser Ala Ser Asp Leu Glu Thr Ser
Gly Leu Gln Glu Gln Arg Asn 35 40 45His Leu Gln Gly Lys Leu Ser Glu
Leu Gln Val Glu Gln Thr Ser Leu 50 55 60Glu Pro Leu Gln Glu Ser Pro
Arg Pro Thr Gly Val65 70 75432PRTHomo sapiens 4Ser Pro Lys Met Val
Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp1 5 10 15Arg Ile Ser Ser
Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30
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